Novel thioredoxin inhibitors paradoxically increase hypoxia-inducible factor-α expression but decrease functional transcriptional activity, DNA binding, and degradation

Purpose: Hypoxia-inducible factor-alpha (HIF-alpha) is a transcription factor that regulates the response to hypoxia. HIF-alpha protein is found at high levels in many cancers, and the redox protein thioredoxin-1 (Trx-1) increases both aerobic and hypoxia-induced HIF-alpha. Therefore, Trx-1 and HIF-alpha are attractive molecular targets for novel cancer therapeutics. Experimental Design: We investigated whether two novel anticancer drugs AJM290 and AW464 (quinols), which inhibit Trx-1 function, can inhibit the HIF pathway. Results: Treatment of several cancer cell lines with AJM290 or AW464 prevented the hypoxia-induced increase of vascular endothelial growth factor (VEGF) at subtoxic concentrations. AJM290 and AW464 also decreased VEGF in pVHL mutant renal cell carcinoma cells that constitutively overexpress HIF-alpha protein. They surprisingly up-regulated HIF-alpha expression in breast cancer cell lines in normoxia and hypoxia as well as in pVHL mutant cells. In the MDA-MB-468 breast cancer cell line, the compounds inhibited RNA and protein expression of the HIF-alpha target genes, carbonic anhydrase IX, VEGF, and BNIP3, concordantly with HIF-alpha up-regulation. Both compounds specifically inhibited HIF-alpha-dependent induction of hypoxia regulatory element-luciferase and HIF-1 alpha hypoxia regulatory element-DNA binding. To analyze the HIF-1 alpha domain inhibited by AJM290, we transfected cells with plasmids expressing a fusion protein of Gal linked to HIF-1 alpha or HIF-1 alpha COOH-terminal transactivation domain (CAD) with a Gal4-responsive luciferase reporter gene. AJM290 inhibited both the full-length HIF-1 alpha and HIF-1 alpha CAD transcriptional activity. Conclusions: AJM290 and AW464 are inhibitors of HIF-1 alpha CAD transcription activity and DNA binding, but they also inhibit degradation of HIF, in contrast to other Trx inhibitors.